The central hypothesis of this application is that oncogenic PI3K/Akt signaling drives metabolic reprogramming to promote breast tumor initiation and progression, resulting in cancer-specific metabolic vulnerabilities that are therapeutically tractable. While there has been much interest in understanding how this pathway contributes to aerobic glycolysis in cancer, mechanisms by which PI 3-K/Akt signaling modulates other metabolic processes to synthesize metabolites required for tumor growth are not well defined. Using robust models for studying PI 3-K/Akt signaling in breast cancer, we propose a project to evaluate the metabolic changes mediated by PI3K/Akt to promote tumor initiation and progression, with a focus on two antioxidant pathways: (i) the synthesis of glutathione (GSH), the major cellular antioxidant, and (ii) the synthesis of cysteine, which is involved in multiple antioxidant systems, through the transsulfuration pathway. In Aim 1, we will extend our preliminary studies by evaluating the mechanisms by which oncogenic PI3K and Akt regulate GSH biosynthesis to modulate the cellular redox state. We will focus on the activation of Nrf2, a key transcription factor in the antioxidant defense system, as a major mechanism downstream of PI3K/Akt in GSH biosynthesis. We will evaluate the requirement for GSH biosynthesis in tumor initiation mediated by oncogenic PI3K/Akt and identify therapeutic strategies that exploit GSH dependence in tumor maintenance. In Aim 2, we will investigate the metabolic determinants for Akt2 specificity in the context of PTEN inactivation, with a focus on antioxidant metabolism. We will perform targeted metabolomics in PTEN-deficient cell lines coupled with SILAC phospho-proteomics to identify specific targets of Akt2, with prioritization focused on metabolic enzymes. We will also investigate the mechanistic basis for isoform-specific Akt2 substrate selection. These substrates may define potential therapeutic targets or biomarkers to guide specific therapies. In Aim 3, preliminary data indicate that a subset of breast cancer cells preferentially shunt the metabolite homocysteine away from methionine synthesis via the methionine cycle and towards the production of cysteine through the transsulfuration pathway. Cysteine, in turn, is involved in multiple antioxidant systems, including the synthesis of GSH. Oncogenic Akt is sufficient to confer this phenotype. We will assess how PI3K/Akt regulates transsulfuration pathway genes and assess pathway activity by metabolic analyses. Finally, we will evaluate the transsulfuration pathway genes CBS and CTH as potential therapeutic targets in breast cancer. Identifying these mechanisms as critical determinants for initiation and progression of breast cancers addicted to oncogenic PI3K/Akt will spur development of new antagonists to target antioxidant metabolism through GSH biosynthesis and the transsulfuration pathway. Our findings will provide an integrated, mechanistic understanding of how oncogenic signaling interfaces with metabolic reprogramming and expose cancer-specific metabolic vulnerabilities that constitute new therapeutic opportunities for breast cancer.